Titanium dioxide (TiO2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.

We have synthesized functional nanocomposites consisting of titanium dioxide (TiO 2 ) nanoparticles covalently bound to DNA oligonucleotides. The TiO 2 nanoparticle is approximately 4.5 nm in diameter and retains photocatalytic properties when bound to DNA via dopamine [1]. The DNA attached to TiO 2 also maintains its ability to bind complimentary DNA sequences. Excitation of these nanocomposites by electromagnetic radiation (above 3.2 eV) leads to accumulation of electropositive holes in the bound DNA causing cleavage [1]. We hypothesize that these novel nanocomposites will be capable of targeting sequence specific aberrant and foreign DNA which cause human disease (oncogenes, retroviruses, and genomes of endoparasites). In the present study we used optical microscopy and X-ray fluorescence microscopy to show that it is possible to achieve directed subcellular localization of the TiO 2 -DNA nanocomposites depending on the sequence of DNA bound to the nanoparticle.Oligonucleotides complimentary to genomic DNA encoding 18S rRNA (ttccttggatgtggt) were synthesized to be 3' conjugated to fluorescent molecule tetramethylrhodamine (TAMRA). Such fluorescent oligonucleotides were bound to TiO 2 nanocomposites as described [1]. Human cells with 2n chromosomes contain approximately 300 copies of 18S rRNA gene which are located in the nucleolus [2], while polyploidy cancer cell lines may contain even more copies of this gene in, frequently, several nucleoli. Rat pheochromocytoma PC12 cells at 60-80% confluence were transfected (Superfect, Qiagen) with the TiO 2 -R18S(TAMRA) nanocomposites according to the manufacturer's recommendations. The cells were then washed with PBS, scraped, and pelleted (2500rpm). The cell pellets were resuspened in 10-20µl of F12K media supplemented with 10% serum and seeded on formvar coated EM grids. Cells were allowed to attach to the EM grids for 2-3 hours and then fixed in cold 100% methanol at -20˚C. These cells were stained with Syto RNASelect green fluorescent cell stain to label the nucleolus (Molecular Probes S32703) and Hoechst (Molecular Probes, H3570). The cells were then visualized with the Zeiss LSM 510 Laser Scanning Confocal Microscope for the location of TAMRA labeled oligonucleotides. Upon completion of microscopy, the EM grids with the cells were washed from the mounting media by immersion in PBS for 10 minutes and then dehydrated in 100% ethanol for 10 minutes more. The same dried whole cells were then analyzed for the presence of titanium using the 2-ID-D X-ray beamline of the Advanced Photon Source at Argonne National Laboratory [1]. Figure 1 shows there is weak but detectable titanium X-ray fluorescence (Fig. 1B) that overlaps with the presence of fluorescent TAMRA signal in the cells transfected with TiO 2 -R18S(TAMRA) (Fig.